Project Summary/Abstract The goal of the proposed research is to determine how subsets of myeloid dendritic cells (DCs) in mice conditioned with total lymphoid irradiation (TLI) and anti-T cell antibodies (anti-thymocyte serum; ATS) interact with natural killer T (NKT) cells and Treg cells to promote tolerance to combined organ and bone marrow transplants . Our previous studies showed that stable mixed chimerism and tolerance in this model system are dependent on the presence of both type I invariant NKT cells and the CD8+ antigen cross priming subset of DCs, since tolerance observed in wild type mice is abrogated in Jalpha18-/- and Batf3-/- recipients. In addition, the conditioning regimen changes the balance of immune cells to favor the CD8+ DCs and NKT cells, and results in their activation. Studies are designed to elucidate the changes in surface markers, cytokine secretion patterns, gene expression and in vitro and in vivo functions of the DC subsets and NKT cells in recipients after conditioning, and how these changes are required for the interactions between the DCs and NKT cells that promote tolerance. We will test the hypothesis that the interactions require DC stress protein recognition by NKG2D receptors on NKT cells that lead to NKT cell stimulation of Treg cells and myeloid derived suppressor cells (MDSCs) in an IL-4 dependent manner. We will also test the mechanism by which myeloid DCs in TLI- conditioned kidney transplant patients mediate suppression of T cell activation, including changes in gene expression and cytokine secretion. Since we hypothesize that the activated immunosuppressive cells allow for the acceptance of donor hematopoietic progenitors, chimerism, and associated clonal deletion, these studies should provide important new information that impacts on current clinical trials of tolerance in HLA mismatched kidney and hematopoietic cell transplant recipients using TLI based conditioning.